PROJECT SUMMARY/ABSTRACT: The hepatic endoplasmic reticulum (ER)-anchored monotopic proteins, cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content via increased formation or loss via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions (DDIs). DDIs often stem from altered P450 ER-associated degradation (ERAD) elicited by drug-mediated P450 stabilization i.e. ethanol (EtOH), or enhanced drug-mediated P450 degradation (i.e. grapefruit juice). Hepatic P450 ERAD involves two major pathways: Ubiquitin (Ub)-dependent proteasomal degradation (UPD) and autophagic-lysosomal degradation (ALD). Some P450s (CYP3A4, the major human liver/intestinal P450) incur UPD, others (CYP2B1) incur ALD and yet others (EtOH-metabolizing CYP2E1) incur both. The determinants of this differential P450 proteolytic sorting are unknown and their identification are major goals of our future research. Plausible determinants include (i) P450-homomerization in the ER-membrane; (ii) localization in lipid-disordered (ld) versus lipid- ordered (lo; lipid rafts) ER-microdomains, resistant to detergent extraction (DRMs); (iii) propensity for ER or cytoplasmic P450 aggregation and subsequent recruitment by the autophagic receptors p62/Sequestosome and NBR-1 (neighbor of Braca 1 gene); (iv) specific post-translational modifications other than ubiquitination (i.e. phosphorylation, acetylation); and (v) specific structural domains that confer differential sorting into ALD versus UPD to two closely related orthologous or isoformic P450s. We propose to employ various experimental approaches such as: Confocal fluorescence microscopy, bimolecular fluorescence complementation, fluorescence resonance energy transfer (FRET), in-cell chemical crosslinking, rigorous affinity immunopurification (AIP) with alpaca nanobodies, proteomic (LC-MS/MS) analyses, LC-MS/MS analyses of protein interactions and interactant identification through proximity labeling, as well as post-translational modifications, p62-/NBR-1-deletion mutants and gene ablated cells, P450-chimeras and fusion proteins, and relevant genetic (ATG5-/-, p62-/-, NBR-1-/-) mouse models primary cultured rat and human hepatocytes and cell lines. Elucidation of these fundamental aspects of P450 ERAD processes, we believe, are important because they would not only advance our understanding of basic P450 biology/physiology, but also critically impact on P450-dependent therapeutics and pathophysiology, and thus are clinically relevant. Understanding the molecular determinants of P450 levels is critical for precision dosing of P450 drug substrates and for unraveling the role of endogenous P450 substrates in physiology and pathophysiology. We believe the insights gained from these studies will be universally applicable to other cellular proteins.